Dataset Information

Automated annotation of developmental stages of Drosophila embryos in images containing spatial patterns of expression.

ABSTRACT:

Motivation

Drosophila melanogaster is a major model organism for investigating the function and interconnection of animal genes in the earliest stages of embryogenesis. Today, images capturing Drosophila gene expression patterns are being produced at a higher throughput than ever before. The analysis of spatial patterns of gene expression is most biologically meaningful when images from a similar time point during development are compared. Thus, the critical first step is to determine the developmental stage of an embryo. This information is also needed to observe and analyze expression changes over developmental time. Currently, developmental stages (time) of embryos in images capturing spatial expression pattern are annotated manually, which is time- and labor-intensive. Embryos are often designated into stage ranges, making the information on developmental time course. This makes downstream analyses inefficient and biological interpretations of similarities and differences in spatial expression patterns challenging, particularly when using automated tools for analyzing expression patterns of large number of images.

Results

Here, we present a new computational approach to annotate developmental stage for Drosophila embryos in the gene expression images. In an analysis of 3724 images, the new approach shows high accuracy in predicting the developmental stage correctly (79%). In addition, it provides a stage score that enables one to more finely annotate each embryo so that they are divided into early and late periods of development within standard stage demarcations. Stage scores for all images containing expression patterns of the same gene enable a direct way to view expression changes over developmental time for any gene. We show that the genomewide-expression-maps generated using images from embryos in refined stages illuminate global gene activities and changes much better, and more refined stage annotations improve our ability to better interpret results when expression pattern matches are discovered between genes.

Availability and implementation

The software package is availablefor download at: http://www.public.asu.edu/*jye02/Software/Fly-Project/.

SUBMITTER: Yuan L

PROVIDER: S-EPMC3892688 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Publications

Automated annotation of developmental stages of Drosophila embryos in images containing spatial patterns of expression.

Yuan Lei L Pan Cheng C Ji Shuiwang S McCutchan Michael M Zhou Zhi-Hua ZH Newfeld Stuart J SJ Kumar Sudhir S Ye Jieping J

Bioinformatics (Oxford, England) 20131203 2

<h4>Motivation</h4>Drosophila melanogaster is a major model organism for investigating the function and interconnection of animal genes in the earliest stages of embryogenesis. Today, images capturing Drosophila gene expression patterns are being produced at a higher throughput than ever before. The analysis of spatial patterns of gene expression is most biologically meaningful when images from a similar time point during development are compared. Thus, the critical first step is to determine th ...[more]

PMID: 24300439

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Project description:Sample Preparation | Four biological replicate of 25 dechorionated, but non-deyolked, zebrafish embryos were collected at 4 time points of embryonic development: 1-cell stage (0-hpf, hour post fertilization), 2-hph, 4-hpf, and 6-hpf. All 16 pooled embryo samples were lysed independently by adding 100 ul of Mammalian lysis buffer and 1x Protease Inhibitor cocktail. Digestion | Samples were reduced and alkylated with TCEP and 2-chloroacetamide. Six volumes of pre-chilled acetone were added, and the precipitation was let to proceed overnight. The precipitated proteins were dissolved in 100 ul of 50 mM TEAB, mass spectrometry grade trypsin (Promega Gold) was added at 1:40 w/w and the digestion was let to proceed at 37oC overnight. Pierce fluorometric peptide assay was performed on 10 ul of each digested sample. Tandem Mass Tag (TMT) Labeling | 20 ul of anhydrous acetonitrile were added to each of the 0.5mg TMTpro 16plex reagents. For each sample, 20 ug of peptides were measured out and adjusted to 100 ul with 100 mM TEAB, then mixed with the TMT reagents. The replicate samples for each of the time points were labeled for 1 hr with TMTpro-126, 127N, 127C, -128N (0-hpf, replicates 1-4); 128C, 129N, 129C, 130C (2-hpf, 1-4); 130C, 131N, 131C, 132N (4-hpf, 1-4), 132C, 133N, 133C, 134N (6-hpf, 1-4), respectively. After checking labeling efficiency (>99%), 30 ul each of the 16 differentially labeled samples were combined in a new tube and the resulting volume was reduced using a SpeedVac concentrator to less than 10 ul. High pH Reverse Phase Fractionation | 300 ul of the TMT-labeled peptide mixture (in 0.1% TFA) were loaded onto a Pierce high pH fractionation cartridge placed on a 2.0 ml sample tube. After centrifuging at 3000 x g for 2 minutes, the eluate was collected as the flow-through fraction. The loaded cartridge was placed on a new sample tube, washed with 300 ul of ddH20, and the eluate collected as wash fraction. An additional round of washing was performed using 300 ul of 5% acetonitrile in 0.1% TFA to remove unreacted TMT reagent. A total of 9 HpH RP fractions (E1-E9) were collected by sequential elution in new sample tubes using 300 ul of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50%, and 80% acetonitrile in 0.1% TFA. Dried samples were resolubilized in 44 ul of buffer A (5% acetonitrile in 0.1% formic acid, FA) before LC-MS analysis. Multiplexed Mass Spectrometry Analysis | TMT-labeled peptides were analyzed on an Orbitrap Eclipse Tribrid Mass Spectrometer with a FAIMS Pro interface, equipped with a Nanospray Flex Ion Source, and coupled to a Dionex UltiMate 3000 RSCLnano System. Peptides were loaded on an Acclaim PepMap 100 C18 trap cartridge (0.3 mm i.d., 5 mm length) with the U3000 loading pump via the autosampler. A 75 um i.d. analytical microcapillary column was packed in-house with 250 mm of 1.9 um ReproSil-Pur C18-AQ resin (Dr. Masch). The Orbitrap Eclipse was set up to run the TMT-SPS-MS3 method with three FAIMS compensation voltages (CV) at -40V, -55V, and -70V and a cycle time of 1 sec. Briefly, peptides were scanned from 400-1600 m/z in the Orbitrap at 120,000 resolving power before MS2 fragmentation by CID at 35% NCE and detection in the ion trap set to turbo detection. Dynamic exclusion was enabled for 45s. Real time search (RTS) was performed during the ddMS2 IT CID step against a non-redundant Danio rerio sequence database downloaded from UniProtKB 2021-03 and complemented with common contaminants. Carbamidomethyl and TMTpro16plex (304.2071 Da on Kn) were searched statically, while methionine oxidation was searched as a variable modification. Synchronous precursor scanning (SPS) selected the top 10 MS2 peptides for TMT reporter ion detection in the Orbitrap using HCD fragmentation at 65% NCE at 50,000 resolving power. In addition, RTS was also set up to reject further fragmentation of peptides mapping to contaminants and yolk proteins using an exclusion list with the keywords Contaminant, Phosvitin, and Vitellogenin. MS/MS Data Processing | The LC/MSn dataset was searched using Proteome Discoverer 2.4 against the same protein database used for RTS. SEQUEST-HT implemented through Proteome Discoverer was set up as: precursor ion mass tolerance 10 ppm, fragment mass tolerance 0.6 Dalton, up to two missed cleavage sites, static modification of cysteine, and lysine and peptide N-termini with TMT tag (+229.163 Da) and dynamic oxidation of methionine. Results were filtered to a 1% FDR at peptides levels using Percolator through Proteome Discoverer. MS3 spectra were processed to extract intensity for each reporter ion. Proteins were quantified by summing reporter ion intensities across all matching PSMs.

Dataset Information

Automated annotation of developmental stages of Drosophila embryos in images containing spatial patterns of expression.

Motivation

Results

Availability and implementation

Publications

Automated annotation of developmental stages of Drosophila embryos in images containing spatial patterns of expression.

Similar Datasets

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